The Streptomyces viridochromogenes product template domain represents an evolutionary intermediate between dehydratase and aldol cyclase of type I polyketide synthases

The product template (PT) domains act as an aldol cyclase to control the regiospecific aldol cyclization of the extremely reactive poly-β-ketone intermediate assembled by an iterative type I polyketide synthases (PKSs). Up to now, only the structure of fungal PksA PT that mediates the first-ring cyclization via C4–C9 aldol cyclization is available. We describe here the structural and computational characterization of a bacteria PT domain that controls C2–C7 cyclization in orsellinic acid (OSA) synthesis. Mutating the catalytic H949 of the PT abolishes production of OSA and results in a tetraacetic acid lactone (TTL) generated by spontaneous O-C cyclization of the acyl carrier protein (ACP)-bound tetraketide intermediate. Crystal structure of the bacterial PT domain closely resembles dehydrase (DH) domains of modular type I PKSs in the overall fold, dimerization interface and His-Asp catalytic dyad organization, but is significantly different from PTs of fungal iterative type I PKSs. QM/MM calculation suggests that the catalytic H949 abstracts a proton from C2 and transfers it to C7 carbonyl to mediate the cyclization reaction. According to structural similarity to DHs and functional similarity to fungal PTs, we propose that the bacterial PT represents an evolutionary intermediate between the two tailoring domains of type I PKSs.

Introduction, first paragraph: please provide more references for your statements on modular type I PKS and iterative type PKS Introduction, line 75: "PT-specific sequence insertion involved in dimerization". Please indicate this motif in your sequence alignment that the reader can check and see if AviM has this type of sequence or not.  Fig. S8. Therefore, the title of the paper is wrong. If you want to make a strong statement of an "evolutionary Intermediate", then a more detailed bioinformatic analysis is necessary! The supplementary Figure S8 is insufficient.
In the introduction, you should already do a more detailed comparison of PT and DH domains. What are the similarities what are the differences? What is the catalytic mechanism of a DH domain? Which active site residues does a DH domain use? Are these residues the same as the PT domains use or are they different? To guide your reader. Because later it's important to know how DH domains operate. Maybe you want to show in the introduction already present the proposed/in the literature discussed mechanisms for PT and DH domains.
In the introduction, can you please also provide more examples of bacterial PT domains. Are there other examples known? How do those products of those PT domains look like? Is there a similarity between bacterial Pts? Do they catalyze the same mechanism or different cyclization's? This would be important for your discussion at the end, if your results are "general" or "system-specific" QM/MM: TO me it's a bit unclear what we learn from the MM simulations? Can you please clarify and explain more? What was the goal? How do the results compare to previous experiments in the literature?
To me, your Scheme 1 seems to be an oversimplification. Waters must be involved in substrate coordination and proton transfer reactions. I also cannot see those essential water molecules in the Figure 5? Figure 3; The labels in the Figure are hardly visible. An additional panel showing the residues of the binding site would be helpful for understanding. To me, it seems that CurF and AviM use the same residues for Dimerization. It would also be important to label the residues that form the dimerization site in CurF, AviM and PksA-PT in the sequence alignment (Fig. S1). A superposition of the dimers would be important. Until now it's hard for the reader to understand how different or similar this is. Figure 4: Please indicate the color used also in the legend. The tunnel is hardly visible, can you please use different colors. In general, the tunnels are very specific for the respective substrate and sometimes blocked when no substrate is present, therefore interpretations of Apo-crystal structures must be conservative.  The manuscript entitled "Bacterial Product Template 1 Domain: An Evolutionary Intermediate between Dehydratase and Aldol Cyclase of Type I Polyketide Synthases" by Yuanyuan Feng et al. targeted to characterize the product formation of wt and mutated AviM PT using mass spectrometry methods. Additionally, the authors performed X-ray crystallography to determine the atomic structure of the AviM PT domain, which is similar to dehydrase (DH) domains of modular type I PKSs. The authors also performed QM/MM calculations to demonstrate the mechanism of product formation. This study is performed properly. However, some findings are not very clear. My questions are listed below: Major comments: 1. The authors represented figure 3, the overall structure of AviM PT. and PksA PT. Did the authors calculate the structure of AviM PT and PksA PT? From figure legends, it is very difficult to understand. However, the method shows authors cloned only AviM. If authors used any other published structure, they should mention the PDB ID and write it properly in figure legends.
2. Site-directed mutations were implemented to monitor the product formation. How many new variants do they generate? Are all of them site-directed mutations, or some deletion mutations are also there? If site-directed mutations are implemented, it will be better to underline the gene sequence where the mutation is done. Rewrite the method section correctly.
3. No purification profiles (SEC) and SDS-PAGE are shown for mutated proteins. The size-exclusion chromatography profile for wt AviM PT is very wide ( Figure S5). How pure is the SEC purified protein? No SDS PAGE data showed to assess the quality of the protein. 4. It will be better if the authors represent figure 3A properly. This is the main part of the manuscript. Therefore, it will be better if the authors demonstrate the types of interactions present in the dimer interface of AviM PT? 5. Figure  Minor comments: 1. Line 328 "detectable activity ( Figure 4x)"….. What is figure 4x? 2. In pdf file, Line 229 "E. coli B derived strain developed for the heterologous……", some formatting issues are there. 3. The structure is similar to AviM PT both in overall structure and in organization of His-Asp ""catalytic dyad"" ( Figure S7). However, from figure S7, differences & similarity is not very clear. Figure S7A and Figure   Thirdly, is the enzyme mechanism conserved in fungal and bacterial PT domains. A question that is unanswered is, if AviM is an exception or if it is likely that all bacterial PT domains share the same structure/dimerization/properties Response: Thank you very much for reviewing our manuscript. We completely agree with your comments and have done our best to revise the manuscript. the residues of the binding site would be helpful for understanding. To me, it seems that CurF and AviM use the same residues for Dimerization. It would also be important to label the residues that form the dimerization site in CurF, AviM and PksA-PT in the sequence alignment (Fig. S1). A superposition of the dimers would be important. Until now it's hard for the reader to understand how different or similar this is.

Response:
We adjusted the transparency of labels to make it more clearly. An additional panel C showing interface residues of AviM PT was added in Figure 3. Both AviM PT and CurF DH dimerize via β-sheets of N-terminal hotdog which was shown in Figure S7. The dimer interface residues of AviM PT, CurF DH and PksA PT are labelled in Figure S1 with orange, purple, and green circles, respectively. We superposed CurF DH and PksA PT onto AviM PT in Figure S7 and Figure S9 respectively. Response: Figure S4 was renumbered as Figure S7. The dimeric structures of AviM PT and CurF DH were superposed.
13：Fig S8 The phylogenetic tree is important, maybe you want to include it in the main text as a subpanel. It shows that bacterial PTs are more related to bacterial dehydratases than to fungal PTs, in line with your structural data.

Response:
We put the phylogenetic tree in the main text as the Figure 6.  Table 2 to elucidate the anomalous data quality, which calculated with SHELXD in CCP4. A common name was used for datasets. hours. All primers with underlined mutation sites were listed in Table S1. All plasmids were confirmed by DNA sequencing." (Lines 138-143).
3： No purification profiles (SEC) and SDS-PAGE are shown for mutated proteins.
The size-exclusion chromatography profile for wt AviM PT is very wide ( Figure S5).
How pure is the SEC purified protein? No SDS PAGE data showed to assess the quality of the protein.

Response:
We prepared a new Figure S5 to show SEC and SDS-PAGE profiles of AviM and AviM H949A mutant that were purified for in vitro assays. Additionally, we supplemented the SDS-PAGE profile of AviM PT domain in the Figure S8.  Figure   S7C was prepared to show CurF interface residues (lines 336-338) while Figure S9B was prepared to show PksA PT interface residues (lines 339-343).  Figure S7) was prepared to show structure differences between CurF DH and AviM PT. The figure is shown at different orientation and the differences are highlighted ( Figure S7A). The relative orientation of the two monomers was compared in Figure S7B. The dimer interface of CurF DH was shown in Figure S7C.

Figure
Minor comments: